The present invention provides novel compounds that bind to and activate the erythropoietin (EPO) receptor or otherwise act as EPO agonists. The invention additionally relates to methods for synthesizing the novel compounds, methods of using the novel compounds, and pharmaceutical compositions containing a compound of the invention as the active agent. The invention has application in the fields of biochemistry and medicinal chemistry and particularly provides EPO agonists for use in the treatment of human disease.
Erythropoietin (EPO) is a glycoprotein hormone with 165 amino acids, 4 glycosylation sites on amino-acid positions 24, 38, 83, and 126, and a molecular weight of about 34,000. It is initially produced as a precursor protein with a signal peptide of 23 amino acids. EPO can occur in three forms: xcex1, xcex2, and asialo. The xcex1 and xcex2 forms differ slightly in the carbohydrate components, but have the same potency, biological activity, and molecular weight. The asialo form is an xcex1 or xcex2 form with the terminal carbohydrate (sialic acid) removed. The DNA sequences encoding EPO have been reported. See, Lin (1987) U.S. Pat. No. 4,703,008.
EPO stimulates mitotic division and the differentiation of erythrocyte precursor cells and thus ensures the production of erythrocytes. It is produced in the kidney when hypoxic conditions prevail. During EPO-induced differentiation of erythrocyte precursor cells, there is induction of globin synthesis and increases in the synthesis of the heme complex and in the number of ferritin receptors. This makes it possible for the cell to take on more iron and synthesize functional hemoglobin. Hemoglobin in mature erythrocytes binds oxygen. Thus, the erythrocytes and the hemoglobin contained in them play a key part in supplying the body with oxygen. The complex processes which have been described are initiated by the interaction of EPO with an appropriate receptor on the cell surface of the erythrocyte precursor cells. See, e.g., Graber and Krantz (1978) Ann. Rev. Med. 29:51-66.
EPO is present in very low concentrations in plasma when the body is in a healthy state wherein tissues receive sufficient oxygenation from the existing number of erythrocytes. This normal low concentration is enough to stimulate replacement of red blood cells which are lost normally through aging.
The amount of EPO in the circulation is increased under conditions of hypoxia when oxygen transport by blood cells in the circulation is reduced. Hypoxia may be caused by loss of large amounts of blood through hemorrhage, destruction of red blood cells by over-exposure to radiation, reduction in oxygen intake due to high altitudes or prolonged unconsciousness, or various forms of anemia. In response to tissues undergoing hypoxic stress, EPO will increase red blood cell production by stimulation of proliferation of erythroid progenitor cells. When the number of red blood cells in circulation is greater than needed for normal tissue oxygen requirements, EPO in circulation is decreased.
Because EPO is essential in the process of red blood cell formation, the hormone has potentially useful applications in both the diagnosis and the treatment of blood disorders characterized by low or defective red blood cell production. Recent studies have provided a basis for the projection of efficacy of EPO therapy in a variety of disease states, disorders, and states of hematologic irregularity, including: beta-thalassemia (see, Vedovato et al. (1984) Acta. Haematol. 71:211-213); cystic fibrosis (see Vichinsky et al. (1984) J. Pediatric. 105:15-21; pregnancy and menstrual disorders (see Cotes et al. (1993) Brit. J. Obstet. Gyneacol. 90:304-311; early anemia of prematurity (see Haga et al. (1983) Acta Pediatr. Scand. 72:827-831); spinal cord injury (see Claus-Walker et al. (1984) Arch. Phys. Med. Rehabil. 65:370-374); space flight (see Dunn et al. (1984) Eur. J. Appl. Physiol. 52:178-182); acute blood loss (see Miller et al. (1982) Brit. J. Haematol. 52:545-590); aging (see Udupa et al. (1984) J. Lab. Clin. Med. 103:574-580 and 581-588 and Lipschitz et al. (1983) Blood 63:502-509; various neoplastic disease states accompanied by abnormal erythropoiesis (see Dainiak et al. (1983) Cancer 5:1101-1106 and Schwartz et al. (1983) Otolaryngol. 109:269-272); and renal insufficiency (see Eschbach et al. (1987) N. Eng. J. Med. 316:73-78).
Purified, homogeneous EPO has been characterized. See Hewick, U.S. Pat. No. 4,677,195. A DNA sequence encoding EPO was purified, cloned and expressed to produce synthetic polypeptides with the same biochemical and immunological properties. A recombinant EPO molecule with oligosaccharides identical to those on the natural material has also been produced. See, Sasaki et al. (1987) J. Biol. Chem. 262:12059-12076.
Despite the availability of purified recombinant EPO, little is known concerning the mechanism of EPO-induced erythroblast proliferation and differentiation. The specific interaction of EPO with progenitors of immature red blood cells, platelets, and megakaryocytes remains to be characterized. This is due, at least in part, to the small number of surface EPO receptor molecules on normal erythroblasts and on the erythroleukemia cell line. See, Krantz and Goldwasser (1984) Proc. Natl. Acad. Sci. USA 81:7574-7578; Branch et al. (1987) Blood 69:1782-1785; Mayeux et al. (1987) FEBS Letters 211:229-233; Mufson and Gesner (1987) Blood 69:1485-1490; Sakaguchi et al. (1987) Biochem. Biophys. Res. Commun. 146:7-12; Sawyer et al. (1987) Proc. Natl. Acad. Sci. USA 84:3690-3694; Sawyer et al. (1987) J. Biol. Chem. 262:5554-5562; and Todokoro et al. (1988) Proc. Natl. Acad. Sci. USA 84:4126-4130.
Cross-linked complexes between radioiodinated EPO and cell surface proteins suggest that the cell surface proteins comprise two polypeptides having approximate molecular weights of 85,000 daltons and 100,000 daltons, respectively. More recently, the two cross-linked complexes have been subjected to V8 protease digestion and have been found to have identical peptide fragments, suggesting that the two EPO-binding polypeptides may be products of the same or very similar genes. See, Sawyer et al. (1988) supra. Most cell surface binding studies, however, have revealed a single class of binding sites, averaging 300 to 600 per cell surface, with a Kd of approximately 800 pM (picomolar). See, Sawyer et al. (1987) Proc. Natl. Acad. Sci. USA 84:3690-3694. However, EPO-responsive splenic erythroblasts, prepared from mice injected with the anemic strain (FVA) of the Friend leukemia virus, demonstrate a high and a low affinity binding site with dissociation constants of 100 pM and 800 pM, respectively. See, Sawyer et al. (1987) J. Biol. Chem. 262:5554-5562 and Landschulz (1989) Blood 73:1476-1478. The DNA sequences and encoded peptide sequences for murine and human EPO receptor proteins have been described. See, D""Andrea et al., PCT Patent Publication No. WO 90/08822 (published 1990).
The availability of cloned genes for the EPO receptor (EPO-R) facilitates the search for agonists and antagonists of this important receptor. The availability of the recombinant receptor protein allows the study of receptor-ligand interaction in a variety of random and semi-random peptide diversity generation systems. These systems include the xe2x80x9cpeptides on plasmidsxe2x80x9d system described in U.S. patent application Ser. No. 778,233, filed Oct. 16, 1991, issued as U.S. Pat. No. 5,270,170, the xe2x80x9cpeptides on phagexe2x80x9d system described in U.S. patent application Ser. No. 718,577, issued as U.S. Pat. No. 5,432,018 and in Cwirla et al., Aug. 1990, Proc. Natl. Acad. Sci. USA 87:6378-6382, the xe2x80x9cencoded synthetic libraryxe2x80x9d (ESL) system described in U.S. patent application Ser. No. 946,239, filed Sep. 16, 1992, which is a continuation-in-part application of Ser. No. 762,522, filed Sep. 18, 1991, now abandoned, the xe2x80x9cvery large scale immobilized polymer synthesisxe2x80x9d system described in U.S. patent application Ser. No.492,462, filed Mar. 7, 1990, now U.S. Pat. No. 5,143,854, and those systems described in PCT patent publication No. 90/15070, published Dec. 13, 1990, U.S. patent application Ser. No. 624,120, filed Dec. 6, 1990, now abandoned, Fodor et al., Feb. 15, 1991, Science 251:767-773, Dower and Fodor, 1991, Ann. Rep. Med. Chem. 26:271-180, and U.S. patent application Ser. No. 805,727, filed Dec. 6, 1991, now U.S. Pat. No. 5,424,186.
Novel peptides that interact with the EPO-R have been discovered, and have been disclosed in U.S. Pat. No. 5,773,569 to Wrighton et al. The Wrighton et al. peptides are generally single polypeptide chains about 10 to 40 amino acids in length, and contain a pair of cysteine residues separated by eight amino acids. These EPO-R-binding peptides were found to bind to or otherwise interact with the EPO-R, and were thus presumed to be useful for studying the biological activities of the EPO-R and for treatment of disease involving a deficiency of EPO. A related patent, U.S. Pat. No. 5,830,851 to Wrighton et al., pertains more specifically to therapeutic applications of the aforementioned peptides, and focuses on the use of the compounds to treat disorders such as end-stage renal failure or dialysis, anemia associated with AIDS, autoimmune diseases, malignancies, and chronic inflammatory disease, beta-thalassemia, cystic fibrosis, spinal cord injury, acute blood loss, aging, and neoplastic disease states accompanied by abnormal erythropoiesis.
There remains a need, however, for highly active compounds that bind very specifically to the EPO-R, both for studies of the important biological activities mediated by the receptor and for treatment of diseases, disorders and conditions associated with an EPO deficiency. There remains a further need for improved synthetic methods for the production of such compounds. The present invention provides such compounds and methods, and also provides pharmaceutical compositions and methods for using the compounds as therapeutic agents.
In one embodiment, the invention provides compounds in the form of peptide dimers that bind to and activate the EPO-R or otherwise behave as EPO agonists. The compounds have a first peptide chain R1 and a second peptide chain R2, wherein R1 and R2 may be the same or different, and are linked through a linking moiety. R1 is approximately 10 to 40 amino acid residues in length and comprises a core sequence of amino acids X3X4X5GPX6TX7X8X9 (SEQ ID NO: 1) wherein each amino acid is indicated by standard one-letter abbreviation, X3 is C or homocysteine (Hoc), X4 is R, H, L or W, X5 is M, F, I or nor-leucine (J), X6 is selected from any one of the 20 genetically coded L-amino acids and J, X7 is W, 1-naphthylalanine (also referred to herein as xe2x80x9c1-Nalxe2x80x9d or B) or 2-naphthylalanine (also referred to herein as xe2x80x9c2-Nalxe2x80x9d or U), X8 is D, E, I, L or V, and X9 is C or Hoc. R2 is also approximately 10 to 40 amino acid residues in length and, similarly, comprises a core sequence of amino acids Xxe2x80x23Xxe2x80x24Xxe2x80x25GPXxe2x80x26TXxe2x80x27Xxe2x80x28Xxe2x80x29 (SEQ ID NO: 2) wherein each amino acid is indicated by standard one-letter abbreviation, Xxe2x80x23 is C or Hoc, Xxe2x80x24 is R, H, L or W, Xxe2x80x25 is M, F, I or J, Xxe2x80x26 is selected from any one of the 20 genetically coded L-amino acids and J, Xxe2x80x27 is W, B or U, X8xe2x80x2 is D, E, I, L or V, and Xxe2x80x29 is C or Hoc. The linker moiety between R1 and R2 is preferably although not necessarily a C1-12 linking moiety optionally terminated with one or two xe2x80x94NHxe2x80x94 linkages and optionally substituted at one or more available carbon atoms with a lower alkyl substituent. In addition, a xcex2-alanine residue may be present between R1 and the linking moiety, or between R2 and the linking moiety, or both. Generally, although not necessarily, either (a) X3 and X9 are linked by a disulfide bond and Xxe2x80x23 and Xxe2x80x29 are linked by a disulfide bond, (b) X3 and Xxe2x80x29 are linked by a disulfide bond and Xxe2x80x23 and X9 are linked by a disulfide bond, or (c) X3 and Xxe2x80x23 are linked by a disulfide bond and Xxe2x80x29 and X9 are linked by a disulfide bond.
More preferably, R1 comprises a core sequence of amino acids YX2X3X4X5GPX6TBX8X9X10 (SEQ ID NO: 3) wherein X2, X6 and X10 are independently selected from any one of the 20 genetically coded L-amino acids, X3 is C or Hoc, X4 is R, H or L, X5 is M, F or I, X8 is D, E, I, L or V, and X9 is C or Hoc, R2 comprises a core sequence of amino acids YXxe2x80x22Xxe2x80x23Xxe2x80x24Xxe2x80x25GPXxe2x80x26TBXxe2x80x28Xxe2x80x29Xxe2x80x210 (SEQ ID NO: 4) wherein Xxe2x80x22, Xxe2x80x26 and Xxe2x80x210 are independently selected from any one of the 20 genetically coded L-amino acids, Xxe2x80x23 is C or Hoc, Xxe2x80x24 is R, H or L, Xxe2x80x25 is M, F or I, X8xe2x80x2 is D, E, I, L or V, and Xxe2x80x29 is C or Hoc, and the dimer contains two disulfide bonds.
In a most preferred embodiment, R1 comprises a core sequence of amino acids X1YX2CX4X5GPX6TBX8CX10X11X12 (SEQ ID NO: 5) wherein X1, X2, X6, X11 and X12 are independently selected from any one of the 20 genetically coded L-amino acids, X4 is R or H, X5 is M or F, X8 is D, E, I, L or V, and X10 is R; R2 comprises a core sequence of amino acids Xxe2x80x21YXxe2x80x22CXxe2x80x24Xxe2x80x25GPXxe2x80x26TBXxe2x80x28CXxe2x80x210Xxe2x80x211Xxe2x80x212 (SEQ ID NO: 6) wherein Xxe2x80x21, Xxe2x80x22, Xxe2x80x26, Xxe2x80x211 and Xxe2x80x212 are independently selected from any one of the 20 genetically coded L-amino acids, Xxe2x80x24 is R or H, Xxe2x80x25 is M or F, X8xe2x80x2 is D, E, I, L or V, and Xxe2x80x210 is R, wherein R1 and R2 are identical; the thiol functionalities of X3 and X9 are linked to form an intramolecular disulfide bond within the R1 and, similarly, the thiol functionalities of X3xe2x80x2 and X9xe2x80x2 are linked to form an intramolecular disulfide bond within R2; the linker comprises xe2x80x94NHxe2x80x94R3xe2x80x94NHxe2x80x94 wherein R3 is lower (C1-6) alkylene substituted with a functional group such as a carboxyl group that enables binding to another molecular moiety (e.g., as may be present on the surface of a solid support), and is optionally substituted with a lower alkyl group; and a xcex2-alanine residue is present between R1 and the linker and/or between R2 and the linker. Optimally, the linker is a lysine residue.
According to some embodiments of this invention, two or more, and preferably between two and six amino acid residues, independently selected from any of the 20 genetically coded L-amino acids or the stereoisomeric D-amino acids, will be coupled to the free (amino) terminus of R1 and/or R2, i.e., the terminus of the peptide chain that is not bound to the linker. In addition, or in the alternative, such amino acid residues will be present at the opposing terminus of R1 and/or R2, i.e., between R1 and/or R2 and the xcex2-alanine group, if one is present, or between R1 and/or R2 and the linker moiety, if no xcex2-alanine group is present. For example, the sequence GG will often be appended to termini of the core sequences for ease in synthesis of the peptides. Other modifications are also possible, including capping or otherwise modifying the free amino terminus of R1 and/or R2, replacement of one or more of the naturally occurring, genetically encoded amino acids with a synthetic amino acid, peptide phosphorylation, modification of an amino acid side chain, and the like. Particularly preferred modification comprises covalent attachment of polyethylene glycol moieties at the N-terminus of both R1 and R2.
The invention also provides a method for synthesizing the aforementioned dimer compounds wherein each of R1 and R2 independently contains a cyclic moiety deriving from an intramolecular disulfide bond formed between two cysteine or homocysteine residues. The method is preferably a one-step oxidation process which preferentially results in the desired compound, i.e., a dimer with each peptide chain containing an intramolecular cyclic structure, and minimizes the less desirable (i.e., less active) products in which disulfide bridges are present linking R1 and R2.
The present invention also provides methods for treating disease involving a deficiency of EPO utilizing the novel compounds of the invention, and further provides pharmaceutical compositions comprising one or more compounds of the invention and a physiologically acceptable carrier.